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Abstract

We report the use of fluorescent nanodiamonds (FNDs) as a photostable fluorescent probe for high resolution saturated excitation (SAX) microscopy. We confirmed that FNDs show a nonlinear fluorescence response under saturated excitation conditions generated by intense excitation light. Using FNDs, we quantified the spatial resolution improvement inherent in SAX microscopy, and experimentally demonstrated the scalability of the spatial resolution of SAX microscopy. The photostability of the FNDs allowed us to perform nanoparticle imaging of a multicolor-stained macrophage cell with a spatial resolution beyond the diffraction limit.

Experimentally measured relationship between FND demodulated fluorescence and excitation intensity. The modulation frequency of the excitation laser was 10 kHz. Dotted lines show the gradients of slopes of 1, 2, 3, and 4, as viewed from the left side of the graph.

Fluorescence images of a single FND with a diameter of approximately 100 nm. The fluorescence images were obtained by demodulation at the fundamental frequency (a, 10 kHz) and the harmonic frequencies (b, 20 kHz), (c, 30 kHz), and (d, 40 kHz).

Fluorescence images of FNDs and mitochondria in macrophages in the focal plane (x-y image). a) Dual-color image constructed with a confocal image of mitochondria and a SAX image of FNDs, b) mitochondria and FNDs imaged simultaneously without SAX, c) confocal and d) SAX image of FNDs after bleaching the mitochondria staining dye. The distribution of mitochondria in a) was obtained by subtracting c) from b).

Fluorescence images of FNDs and mitochondria in macrophages along the optical axis (x-z image). a) Dual-color image constructed with a confocal image of mitochondria and SAX image of FNDs, b) mitochondria and FNDs observed simultaneously without SAX, c) confocal and d) SAX image of FNDs after bleaching the mitochondria staining dye. The distribution of mitochondria in a) was obtained by subtracting c) from b).